Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

Chumsae, Chris; Zhou, Liqiang Lisa; Shen, Yang; Wohlgemuth, Jessica; Fung, Emma; Burton, Randall E.; Radziejewski, Czeslaw; Zhou, Zhaohui Sunny

doi:10.1021/ac502179m

Cited by 31 publications

(22 citation statements)

References 65 publications

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“…It is worth noting that the intrinsic charge heterogeneity of the unmodified antibody contributed to the multiple bands observed for both enzymatic and chemical conjugates. As is well documented in the literature, these antibody charge variants are due to various post‐translational modifications, such as deamidation, oxidation, glycosylation, or other reactive metabolites (51‐59).…”

Section: Resultsmentioning

confidence: 99%

Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

Sadiki

Kercher

et al. 2020

Photochem & Photobiology

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Photosensitizer (PS)–antibody conjugates (photoimmunoconjugates, PICs) enable cancer cell‐targeted photodynamic therapy (PDT). Nonspecific chemical bioconjugation is widely used to synthesize PICs but gives rise to several shortcomings. The conjugates are heterogeneous, and the process is not easily reproducible. Moreover, modifications at or near the binding sites alter both binding affinity and specificity. To overcome these limitations, we introduce convergent assembly of PICs via a chemo‐enzymatic site‐specific approach. First, an antibody is conjugated to a clickable handle via site‐specific modification of glutamine (Gln) residues catalyzed by transglutaminase (TGase, EC 2.3.2.13). Second, the modified antibody intermediate is conjugated to a compatible chromophore via click chemistry. Utilizing cetuximab, we compared this site‐specific conjugation protocol to the nonspecific chemical acylation of amines using N‐hydroxysuccinimide (NHS) chemistry. Both the heavy and light chains were modified via the chemical route, whereas, only a glutamine 295 in the heavy chain was modified via chemo‐enzymatic conjugation. Furthermore, a 2.3‐fold increase in the number of bound antibodies per cell was observed for the site‐specific compared with nonspecific method, suggesting that multiple stochastic sites of modification perturb the antibody–antigen binding. Altogether, site‐specific bioconjugation leads to homogenous, reproducible and well‐defined PICs, conferring higher binding efficiency and probability of clinical success.

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Section: Resultsmentioning

confidence: 99%

Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

Sadiki

Kercher

et al. 2020

Photochem & Photobiology

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“…However, formulation additives may also participate in unanticipated reactions to increase acidic charge variants. Chumsae et al () showed that a citric acid derivative reacted with the N‐terminal of an IgG1 molecule which led to increased acidic species. Citric acid is widely used in formulations to maintain buffer pH at 3–6 as it is considered chemically inert.…”

Section: Upstream Process Strategies For Charge Variants Controlmentioning

confidence: 99%

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Chung

Tian

Tan

et al. 2018

Biotech & Bioengineering

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Controlling the charge profile of therapeutic protein is a critical challenge in the current quality-by-design (QbD) paradigm, throughout all phases of biologics process development (PD): cell line development, upstream cell culture, recovery process, downstream purification, and analytical characterization. Charge variant profiles may influence efficacy and/or lead to unintended side-effects. Thus, maintaining a consistent charge profile is of tremendous importance, and increasingly, researchers have focused efforts toward developing strategies to mitigate variability during cell culture and to improve separation and detection of charge variants. Current understanding of factors affecting charge variant formation during manufacturing remains inadequate, and sometimes, even substantial commitment of resources may still not fully achieve the desired or consistent profiles. As such, this review attempts to provide a comprehensive resource for the biologics community by summarizing the impact of charge variants and CQA management, analytical methods for charge variant detection, as well as strategies in downstream and upstream PD for controlling charge variant profiles.

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“…There was no significant change in free amines of the amine‐MSs in phosphate or acetate buffers for at least four autoclave cycles (Figure ). However, in citrate buffer, there was a loss of ∼7% of amines each cycle; this is attributable to the formation of citric acid anhydride during autoclaving, which forms in acidic citrate solutions and can acylate amine groups …”

Section: Resultsmentioning

confidence: 99%

Autoclave sterilization of tetra‐polyethylene glycol hydrogel biomaterials with β‐eliminative crosslinks

Henise

Yao

Ashley

et al. 2020

Engineering Reports

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Sterilization of degradable polymeric biomaterials intended for injection presents a formidable challenge. Often, either the polymer backbone or labile crosslinks controlling degradation are adversely affected by commonly used sterilization methods. The purpose of this work was to develop an approach to sterilize tetra-polyethylene glycol hydrogel microspheres (MSs) with -eliminative crosslinks that are destined to be carriers for drug delivery. The approach taken was to acidify the medium to compensate for the base-catalyzed cleavage of linkers at high temperatures. We determined that rates of linker cleavage at pH 4 or below were sufficiently slow as to allow autoclaving and showed that precursor amine-derivatized MSs could withstand autoclaving at pH 4 for at least four cycles of 20 minutes each at 121 • C. Thus, amine-MSs need not be prepared aseptically, but instead can be prepared in a low bioburden environment, and then sterilized by autoclaving before drug attachment. K E Y W O R D Sbiodegradable hydrogel, drug delivery, half-life extension, microspheres, tetra-polyethylene glycol INTRODUCTIONWe have developed a general approach for half-life extension of therapeutics, in which a drug is covalently tethered to a long-lived carrier by a linker that slowly cleaves by -elimination to release the native drug (Scheme 1). 1 The cleavage rate of the linker is controlled by the nature of an electron-withdrawing "modulator" (Mod) attached to a carbon containing an acidic C-H bond. After rate-determining proton removal, the intermediate rapidly collapses to provide the free drug. These linkers are not affected by enzymes and are stable for years when stored at low pH and temperature. One carrier we use is a large-pore tetra-polyethylene glycol (PEG) hydrogel polymer. 2-4 These hydrogels-fabricated as uniform 50-μm microspheres (MSs)-are injected subcutaneously (SC) or locally through a small-bore needle where they serve as a depot to slowly release the drug. Importantly, a slower cleaving -eliminative linker is incorporated in crosslinks of these polymers, so gel degradation occurs after drug release. 3,5 Fabrication of tetra-PEG hydrogel MS-drug conjugates is achieved in two stages (Scheme 2). 3,4 Stage 1 involves preparation of amine-derivatized MSs. Here, equimolar amounts of an 8-arm PEG containing 4 amine-and 4 azido-linker end groups and a 4-arm PEG containing cyclooctyne end groups are mixed in a droplet forming device. The azide (A) and cyclooctyne (B) end groups of the PEG prepolymers react within the droplets by strain-promoted alkyne-azide cycloadditions (SPAAC) to form 1,2,3-triazoles and provide homogeneous amine-derivatized tetra-PEG

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Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

Cited by 31 publications

References 65 publications

Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Autoclave sterilization of tetra‐polyethylene glycol hydrogel biomaterials with β‐eliminative crosslinks

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